Antibiofilm Activity of Curcumin and Piperine and Their Synergistic Effects with Antifungals against Candida albicans Clinical Isolates

Background Candidiasis is the common name for diseases caused by yeast of the genus Candida. Candida albicans is one of the most implicated species in superficial and invasive candidiasis. Antifungals, polyenes, and azoles have been used to treat candidiasis. However, due to the development of antifungal resistance, research of natural substances with potential antifungal effects at low concentrations or combined is also a possibility. Methods The broth microdilution method was used to evaluate the antifungal activity. The biofilm formation was assessed using the microtiter plate method. The antibiofilm activities were assessed using micro plaque tetrazolium salt assay (MTT). The combination effect of antifungal with natural substances was made using the checkerboard method. Results Among our isolates, clotrimazole was the most resistant, but amphotericin B was the most effective antifungal. The biofilm was formed by all isolates of C. albicans. Curcumin and piperine displayed antibiofilm activity with minimum biofilm inhibitory concentration (MBIC) and minimum eradicating concentration (MBEC) ranging from 64 to 1024 μg/mL and 256 to 2048 μg/mL. In combination, piperine presented double synergistic effects compared to curcumin with all antifungals tested. Curcumin shows more synergistic effect when combined with polyenes than with azoles. However, piperine shows a more synergistic effect when combined with azoles compared to polyenes. Conclusion C. albicans was susceptible to curcumin and piperine both on planktonic cells and biofilm. The combination of curcumin and piperine with antifungals has shown synergistic effects against multiresistant clinical isolates of Candida albicans representing an alternative drug research for the treatment of clinical candidiasis.


Introduction
Fungal diseases have emerged and have been increasingly recognized as important public health problems owing to an ever-expanding population of immune-compromised patients.Tey are usually mostly caused by Candida species as C. albicans has been reported as the most prevalent pathogen in systemic fungal infections during the last three decades [1].Antifungals are currently used in the treatment of yeast infections.Diferent antifungals are commonly used in therapy and target fungi: chitin synthesis, ergosterol synthesis, glucan synthesis, squalene epoxidase, nucleic acid synthesis, protein synthesis, and microtubule synthesis.Azoles (fuconazole and clotrimazole) are used to treat fungal infection and have fungistatic efects on C. albicans because they inhibit cytochrome P450 14α-lanosterol demethylase and, then, block the synthesis of ergosterol in the cytoplasm.Azoles reduce the amount of ergosterol in the membrane by inhibiting its synthesis in the cytosol.Antifungal polyenes such as amphotericin B and nystatin bind to ergosterol (the major sterol of the fungal membrane) and have fungicidal activities.
So, commercial antifungal agents, including fuconazole and amphotericin are widely prescribed, but they are not very efective in clinical situations [2].Due to the toxicity of commercial antifungals and the multiresistance of C. albicans to antifungals, antifungal therapy to combat candidiasis is still inefective [3].Te pathogenicity of C. albicans increases because of the resistance activity of virulence factors like bioflm formation and yeast-to-hyphae transition [2].Bioflm is defned as a microbial community containing a dense network of yeast and flaments embedded inside an exopolymeric matrix that hinders the action of antimicrobials.It acts as a difusion barrier against antifungals and holds immune factors in comparison to planktonic cells [4].C. albicans bioflm shows increased resistance against most antifungal agents and is difcult to eradicate [2].Te increased cost and drug resistance have put limitations on the use of antifungal drugs, so there is a need to fnd better drug agents to cure life-threatening infections associated with the bioflm of C. albicans [2].
Among the potential sources of new agents, a new strategy consisting of the use of natural products to promote health is as old as human civilization.Recently, it was reported that natural products derived from plants as abundant sources of biologically active compounds have driven their exploitation toward the search for new chemical products that can lead to further pharmaceutical formulations [4].Many studies have reported the in vitro activities of various yeast species.Curcumin, found in Curcuma longa, is an important Asian spice used in many food preparations.Previous studies report that curcumin is a promising anticandida compound of clinical interest [5].Piperine is a naturally occurring alkaloid found in consumed species of black pepper (Piper nigrum) and long pepper (Piper longum) and has antimicrobial and antibioflm activities against bacteria strains [6,7].
Another approach to overcome microbial infections associated with bioflm formation is to use a combination therapy of natural substances with commercial antimicrobial drugs to enhance treatment [7].Combination therapy is considered an efective approach to improving the efcacy of therapy in the treatment of invasive infections.Additionally, combination therapy is very useful and efective since it may increase both the rate and degree of microbial killing because each drug has a diferent mechanism of action [2].Due to diferent targeting approaches, the development of drug resistance can be slowed down, and the liver toxicity of antifungals like fuconazole should be avoided with the help of two or more combined drugs [2].Tis study aimed to evaluate the in vitro antifungal and antibioflm activity of two natural substances, piperine (alkaloid) and curcumin (a polyphenol), and their combination with current antifungals, revealing species with inhibition/reduction efects on the bioflm formation in Candida albicans isolates.

Antifungal Susceptibility.
Te minimum inhibitory concentrations (MICs) of the antifungals and natural products were determined by the method previously described [8].Te natural substances and antifungals were prepared at 4096 μg/mL and 512 μg/mL, respectively, and serially diluted twice with SDB in a 96-well microplate to obtain a fnal volume of 100 μL.Te concentrations of natural substances and antifungals ranged, respectively, from 2048 to 1 μg/mL and 256 to 0.125 μg/mL.Subsequently, 100 μL of fungal inoculum at a concentration of 1.5 × 10 4 CFU/mL was added to the microplate wells and incubated at 37 °C for 48 hours.Wells containing only fungal inoculum represented the negative control; however, wells containing microorganisms and standard drugs were considered the positive control.
After incubation, the MIC endpoint was considered the lowest concentration of natural substances or antifungals where no growth was observed in the microplate.Te use of vital dyes in assessing the antifungal activity of natural substances may compromise the comparability of the data.
Te antifungal activity of natural products was considered as follows: most active (MIC value ≤1 μg/mL), significant activity (1 ≤ MIC value ≤10 μg/mL), moderate (10 ≤ MIC value ≤100 μg/mL), and inactive (100 < MIC value ≤1000 μg/mL) [9].Te cut-of values of antifungals previously described were used for Candida albicans [8].For fuconazole, yeast with a MIC value ≤8 μg/mL was considered susceptible, while yeast with 32 ≥ MIC value ≥32 μg/ mL was considered as intermediate, and yeast with a MIC value ≥64 μg/mL was considered as resistant.For amphotericin B and nystatin, the MIC value ≤ 1 μg/mL indicated that the yeast was susceptible, while yeast with 2 ≥ MIC value ≥4 μg/mL was considered intermediate, and then, MIC (1) 2.5.Bioflm Inhibition Assay.Te bioflm inhibition activity of curcumin, piperine, and antifungals was carried out according to the method previously described [11].Briefy, 20 μL of fungal inoculum (1.5 × 10 4 CFU/mL) and 180 μL of concentrations of antifungals or natural substances were introduced into the microplate.Final concentrations of antifungals and natural products, respectively, range from 8 to 1024 μg/mL and 16 to 2048 μg/mL, and the microplate was incubated at 37 °C for 48 h.Ten, the microplates then carefully cleared of their contents and washed three times with phosphate bufer (PBS), pH 7.2.A volume of 150 μL of methanol was added to the well for bioflm fxation and removed after 15 min, and then 150 μL of crystal violet (1%) was added for staining.Ten, microplates were washed twice with PBS to discharge the stain.After the air-dying process, the dye of bioflms that lined the walls of the microplate was solubilized with 150 μL of 98% ethanol.Ten, the optical density (OD) of the microplate was measured spectrophotometrically at 570 nm by using a microplate reader.Te study was performed three times.Uninoculated well containing sterile RPMI-1640 was used.Te percentage of bioflm was calculated using the formula below, and the minimal bioflm inhibitory concentration (MBIC) was recorded as the lowest concentration of antifungals or natural substances that inhibit 100% of bioflm.

% Biofilm inhibition activity
2.6.Bioflm Eradication Assay.Te determination of the bioflm eradication potential of curcumin, piperine, and antifungals was performed as previously described [11].Briefy, 200 μL of fungal inoculum (1.5 × 10 4 CFU/mL) and 180 μL of RPMI-1640 were introduced into the microplate and incubated at 37 °C for 48 h.Once the bioflm had formed, the microplate well was gently cleared of its contents and washed three times with PBS bufer.Ten, 200 μL of antifungals and natural substances at concentrations ranging from 8 to 1024 μg/mL and 16 to 2048 μg/mL and incubated at 37 °C for 48 hours.After incubation, the microplate was treated as described previously for the bioflm inhibition assay.Te test was repeated three times, and the percentage of bioflm eradication was calculated using the formula below.Te minimal bioflm eradicating concentration (MBEC) was recorded as the lowest concentration of antifungals or natural substances that reduce 100% of bioflm.

Combination of Antifungals with Curcumin and Piperine
against Planktonic C. albicans Isolates.Te checkerboard assay as previously described [12] was used for the determination of the combined efects of antifungals with curcumin and piperine against Candida albicans.Briefy, 50 μL of Sabouraud dextrose broth (SDB) was distributed into each well of the microdilution plates.Antifungals were serially diluted along the abscissa, and natural substances were serially diluted along the ordinate.Piperine in combination with fuconazole showed fve synergistic efects (FIC � 0.15 to 0.5) against C. albicans isolates Ca10, Ca02, Ca14, Ca16, and ATCC 9002 with a reduction of the MIC value of fuconazole (2048-, 256-, 2048-, and 512, respectively).Four synergistic efects were obtained with a combination of piperine and clotrimazole against isolates Ca03, Ca02, Ca14, and ATCC 9002 with FICI values (0.28, 0.37, 0.37, and 0.31, respectively) and reducing MIC values of fuconazole 32-fold, 4-fold, 8-fold, and 16fold.Tree synergistic efects were also reported with piperine and nystatin against isolates Ca16, Ca14, and ATCC 9002, with FIC values ranging from 0.15 to 0.37 and reducing MIC values of nystatin from 8 to 64-fold.Two synergistic efects were shown with piperine and amphotericin B against isolates Ca02 and Ca14 with FICI values 0.25 and 0.5, respectively, and reducing 8-fold and 4-fold the MIC value of amphotericin B.  Generally, in all our isolates and strains, piperine presented a double number (thirteen) of synergistic efects compared to curcumin (six) in combination with all antifungals.Curcumin presented more synergistic efects (four) combined with polyenes (amphotericin B and nystatin) compared to azoles (two) (clotrimazole and fuconazole).However, piperine presented more synergistic efects combined with azoles (nine) compared to polyenes (fve).

Discussion
C. albicans is one of the most common pathogenic fungi in humans, causing superfcial and systemic infections.Te ability of C. albicans to form bioflms makes them resistant and more tolerant to antimicrobial therapy.Given the resistance of C. albicans to antifungal agents as a result of bioflm formation, it is becoming difcult to predict which molecules will emerge as new clinical antifungal agents.Bioflm formation makes treatment difcult and contributes to high rates of morbidity and mortality.Current antifungals are extremely limited, and six classes of antifungal drugs are used to treat fungal infections, namely, azole derivatives, polyenes, echinocandins, 5-fuorocytosine, allylamines, and morpholines [13].
Te antifungal susceptibility of C. albicans against antifungals and natural substances is presented in Table 1.Clotrimazole was the antifungal agent with the highest incidence of resistance.However, amphotericin B was the most efective against C. albicans.Comparatively, previous studies show the MIC values range from 1 to 16 μg/ mL for nystatin and a MIC value of 0.5 μg/mL for amphotericin B [14].Moreover, it was reported that MIC values for clotrimazole ranged from 8 to 16 μg/mL and from 32 to 64 μg/mL.Clotrimazole was reported as the most efective anticandida drug compared to fuconazole and nystatin [15].Our results corroborated those obtained previously.According to the epidemiological cut-of values of antifungals, azoles were more resistant than polyenes.Compounds that act by lysing the membrane have lower resistance rates.Tis is because the modifcations to the plasma membrane induced by the pathogen to become resistant to these compounds normally have a major impact on its viability.For their mechanism of action, azoles (fuconazole and clotrimazole) act on the inhibition of lanosterol 14 α-demethylase (ERG11; ergosterol biosynthesis), and polyenes (nystatin and amphotericin B) bind to ergosterol in the fungal cell membranes; formation of transmembrane pores, resulting in loss of membrane integrity, and interruption of the ion gradient, and disturbing normal membrane function.Tis high resistance of Candida strains to azoles may be caused by drug efux due to a reduction in the afnity of the Erg11 protein through mutations.Mutations in the Erg11 protein also upregulate multiple drug transporter genes.Changes in specifc stages of the ergosterol biosynthesis pathway were seen [13].
Due to the development of the resistant form of Candida albicans, conventional drugs can be sometimes inefective.Herbs and naturally imitative bioactive compounds could be a new source of antimycotic therapy.Several review studies suggest that herbal medicines and natural bioactive compounds have antifungal efects [16].Nutraceuticals such as curcumin (Curcuma longa, polyphenol) and piperine (Piper nigrum and Piper longum an alkaloid) are useful in the treatment of C. albicans in candidiasis and could be a safe, accessible, and inexpensive management option to prevent and treat disease [16,17].
Te anti-C.albicans susceptibility to curcumin and piperine was evaluated and presented in Table 1.Curcumin presented signifcant and moderate activities.Moreover, piperine shows moderate activity on C. albicans isolates.Our results corroborate the previous studies reporting that curcumin and piperine were inactive against the majority of C. neoformans fungus isolates with MIC values of more than 100 μg/mL.Compounds with a lytic action on the membrane have a better antibioflm efect.Curcumin, which acts by lysing the fungal cell, has a more powerful antibioflm efect than piperine [17].
Te bioflm formation enhances tolerance to antifungal drugs among Candida species and has necessitated the search for a new antifungal treatment strategy.Interference in pathogenic bioflm development by new antifungal compounds is considered an attractive antiinfective strategy [18].Tis study evaluated the bioflm's abilities compared to the reference strain ATCC 9002 presented in Figure 1.Te results showed that all our isolates formed bioflm at 48 hours with diferent percentages.Te percentages of bioflm in the isolates were higher than for the reference strain ATCC 9002.Te ability of C. albicans to switch morphology and form bioflms is the central property of Scientifca their pathogenesis.Because bioflms formed by C. albicans are inherently tolerant of immune systems and conventional antifungals, and therefore, their susceptibility to current therapeutic agents remains low [19].
In the present study, a plant-derived alkaloid, piperine, polyphenol, and curcumin, were investigated for antibioflm activity against C. albicans and presented in Table 2. Curcumin and piperine were efective against C. albicans bioflms.However, curcumin showed better activity against C. albicans bioflm compared to piperine.According to the R (MBEC/MBEC) ratio, the concentration of antifungal or natural substances for inhibition was lower than that of the eradicated bioflm of Candida albicans.In fact, by their mechanism of action, curcumin binds to ergosterol present in the membrane, which leads to fungal cell disruption and loss of intracellular content [20].Piperine signifcantly downregulates the expression of several bioflm-related and hyphal-specifc genes (ALS3, HWP1, EFG1, and CPH1) [21].
In addition to complete inhibition and eradication of bioflm, another strategy is to fnd combinations of compounds with anticandida activity [2].Te efect of the combination of antifungals and natural substances was evaluated, and the results are presented in Table 3.Our results showed that curcumin and piperine enhanced the activities of antifungals and presented a synergistic efect against C. albicans.
Curcumin combined with nystatin showed three synergistic efects against C. albicans strains, reducing 4-fold, 64-fold, and 4-fold, respectively, the MIC of nystatin.Moreover, combined with amphotericin B, it showed three synergistic efects, reducing 4-fold the MIC of amphotericin B. Two synergistic efects were also obtained with a combination of curcumin and fuconazole, reducing 8-fold and 32-fold the MIC of fuconazole.Ten, combined with amphotericin B, it showed one synergistic efect and reduced 4-fold the MIC value of amphotericin B. No synergistic efects were obtained with a combination of curcumin and clotrimazole.Our results corroborated the previous studies, which reported the synergistic efect of all combinations of curcumin and amphotericin B, whereas both synergistic and additive efects were observed in the combination of curcumin and fuconazole, suggesting that these combinations should provide greater fungicidal efects for the treatment of systemic and superfcial candidiasis [22].
As concerning piperine, in combination with fuconazole, it showed fve synergistic efects against C. albicans isolates.Four synergistic efects were obtained with a combination of piperine and clotrimazole, with FICI values of 0.28, 0.37, 0.37, and 0.31, respectively, and reducing MIC values of fuconazole 32-fold, 4-fold, 8-fold, and 16-fold.Tree synergistic efects were also reported with piperine and nystatin against isolates with FICI values ranging from 0.15 to 0.37 and reducing MIC values of nystatin from 8 to 64-fold.Two synergistic efects were shown with piperine and amphotericin B with FICI values of 0.25 and 0.5, respectively, and reducing the MIC value of amphotericin B. In the same idea, the synergistic efect of the combination of piperine with azoles (ketoconazole) was previously reported against C. albicans [23].Among all our isolates and strains, piperine presented a double number (thirteen) of synergistic efects compared to curcumin (six) in combination with all antifungals.Curcumin presented more synergistic efects (four) combined with polyenes (amphotericin B and nystatin) compared to azoles (two) (clotrimazole and fuconazole).However, piperine presented more synergistic efects combined with azoles (nine) compared to polyenes (fve).
Te limitations of this study are threefold: frstly, we did not evaluate the mechanism of action at the molecular level of our synergistic combinations on the bioflm extracellular matrix.Furthermore, we did not evaluate the efect of the combinations on quorum sensing inhibition, a signaling mechanism that bacteria within the bioflm use to enhance their pathogenicity.Finally, in this study, synergistic combinations were obtained only in vitro and were not evaluated in vivo.
Overall, the diference between this study from similar ones lies in its comprehensive exploration of both curcumin and piperine, as well as their synergistic efects with antifungals.While previous studies have focused on other bacterial species, the inclusion of both curcumin and piperine in this research adds a layer of complexity that mirrors the potential multifaceted nature of combating C. albicans infections.Tis comprehensive approach enhances the translational potential of the study's fndings, ofering a more holistic strategy for clinicians and researchers to consider in the development of antifungal therapies.In summary, this study's strength lies in its unique focus on the synergistic potential of curcumin and piperine with antifungals against multiresistant C. albicans clinical isolates.Te comprehensive exploration of these natural compounds and their combined efects sets this research apart from similar studies, providing a promising avenue for the development of innovative and efective antifungal strategies in clinical settings.

Conclusion
Candidiasis is a major life-threatening disease due to the increased incidence of drug resistance in Candida spp.and the limited antifungals available.C. albicans isolates were mostly resistant to azole antifungals compared to polyenes.Curcumin and piperine showed, respectively, signifcant and moderate activity against planktonic C. albicans.Te resistance of C. albicans was mostly associated with bioflm formation.Antibioflm and combination therapy may be a valid alternative.Natural substances curcumin and piperine showed antibioflm activity, inhibition, and eradication of bioflmmultiresistant C. albicans isolates.Te combination therapy showed a synergistic interaction between curcumin and piperine with antifungal polyenes and azoles against resistant C. albicans.Tere are many reports available on the combination of antifungal drugs with synthetic small molecules and with natural compounds in vitro.Some combinations were tested in vivo.Tere is a need to try these combinations in vivo.

8
Scientifca Scientifcaand the well was incubated at 37 °C for 48 h.Te fnal concentration ranges from 0.25 to 256 μg/mL for antifungals, 4-512 μg/mL for curcumin, and 8-512 μg/mL for piperine.After incubation, a volume of 40 μL of INT (iodonitrotetrazolium chloride) was added to microplate wells and incubated at 37 °C for 30 minutes.Viable fungal cells change the yellow dye of INT to a pink color.
Natural Substances and Antifungals.Te susceptibility profle of C. albicans planktonic cells to antifungals (amphotericin B, nystatin, clotrimazole, and fuconazole) and natural substances is shown in Table1.MIC values range from 0.125 to 256 μg/mL and from 32 to 1024 μg/mL for curcumin and piperine, respectively.Te minimum inhibitory concentration values of antifungals ranged from 0.125 to 64; 0.25 to 128; 0.125 to 64; and 0.5 to 128 μg/mL, respectively, for antifungals: amphotericin B, nystatin, clotrimazole, and fuconazole.According to the epidemiological cut-of values of antifungals, azoles (clotrimazole and fuconazole) were more resistant than polyenes (nystatin and amphotericin B). reference strain ATCC 9002 and presented in Figure1.Te results showed that all our isolates formed bioflm at 48 hours with diferent percentages.Te percentages of bioflm in the isolates (Ca04, Ca08, Ca10, Ca13, Ca14, Ca16, and Ca17) were more than for the reference strain ATCC 9002.Te isolates Ca02, Ca03, Ca10, Ca13, Ca14, and Ca16 presented a percentage of bioflm of more than 50%, and

Table 1 :
Minimum inhibitory concentrations (MICs) of antifungals and natural substances against C. albicans strains.

Table 2 :
Antibioflm activities of natural substances and antifungals against Candida albicans.

Table 3 :
Minimum inhibitory concentration (MIC) and efects of the combination of curcumin/piperine with amphotericin B, nystatin, clotrimazole, and fuconazole on C. albicans isolates.